1. Field of the Invention
The present invention relates to a total internal reflection fluorescence microscope for performing fluorescence observation by use of an evanescent light generated by total internal reflection illumination.
2. Description of the Related Art
In recent years, a total internal reflection fluorescence microscopy (hereinafter referred to as TIRFM) has attracted attentions as a microscope for fluorescence observation of a living thing. In this TIRFM, an illuminative light is totally reflected by a boundary surface between a cover glass and a specimen, and a fluorescent substance is excited using a light called an evanescent light which leaks into a small region having a size of several hundreds nm or less on a specimen side. In this TIRFM, only the fluorescence of the small region in the vicinity of the cover glass is observed. An observed image of TIRFM provides a very dark background. Accordingly, it is possible to observe fluorescence having a high contrast and faint fluorescence.
Additionally, in a site of biological research using TIRFM, there are a case where a shallow plane is to be observed with good contrast in the vicinity of the boundary surface between the cover glass and the specimen, and a case where the evanescent light is extended to a certain degree of depth to observe a broad range. Therefore, it is desirable to change a leak-out depth of the evanescent light in accordance with the specimen.
The leak-out depth of the evanescent light from the boundary surface is described, for example, in D. Axelrod's document “Total Internal Reflection Fluorescence at Biological Surfaces”. Accordingly, the following equation is established.d=λ/4π√{square root over ((n12·sin θ12−n22))}  (1),where d denotes the leak-out depth of the evanescent light, λ denotes a wavelength of the light, n1 denotes a refractive index on the incidence side, θ1 denotes an incidence angle, and n2 denotes a refractive index on an emission side.
Therefore, when the incidence angle θ1 of the illuminative light with respect to the boundary surface, that is, an inclination angle of the illuminative light with respect to a normal to the boundary surface increases, the leak-out depth d of the evanescent light becomes shallow. In actual TIRFM, a laser beam having a high coherent property is used, and the incidence angle of the illuminative light is adjusted. Accordingly, the incidence angle of the laser beam onto the boundary surface changes, and the leak-out depth of the evanescent light is adjusted.
FIGS. 17A to 17C are diagrams showing a function of TIRFM described in Jpn. Pat. No. 3093145. An objective lens 1 has a numerical aperture with which total internal reflection illumination is possible. A specimen 3 is laid on a cover glass 2. A mirror 4 is movable in a direction crossing an optical axis direction of the objective lens 1 at right angles.
A laser beam 5 for use as the illuminative light is incident upon the mirror 4. In this state, as shown in FIGS. 17A to 17C, the mirror 4 moves in the direction crossing the optical axis direction of the objective lens 1 at right angles. Accordingly, an incidence position of the laser beam incident upon the objective lens 1 moves in a direction distant from an optical axis of the objective lens 1. By the movement of the incidence position of the laser beam, the incidence angle of the laser beam 5 emitted toward the boundary surface between the cover glass 2 and the specimen 3 from the objective lens 1 changes. The laser beam 5 emitted from the objective lens 1 is totally reflected by the boundary surface between the cover glass 2 and specimen 3 via an immersion oil 6 as shown in FIG. 17C.
FIG. 18 is a constitution diagram of TIRFM described in Jpn. Pat. Appln. KOKAI Publication No. 2001-013413. The TIRFM includes a laser illuminating device 7 which outputs the laser beam 5. In the TIRFM, the laser beam 5 is incident upon a side surface 10 of a point ball lens 9 of a condenser lens for transmission illuminating 8 and the total internal reflection illumination is possible. The laser illuminating device 7 is rotatable with respect to a microscope main body 11. The laser illuminating device 7 rotates centering on an intersection of the boundary surface of the cover glass 2 and specimen 3 and the observation optical axis. Accordingly, the laser beam 5 changes its incidence angle with respect to the boundary surface between the cover glass 2 and specimen 3.
Additionally, the incidence angle of the laser beam 5 needs to be inclined by a critical angle or more, at which the total internal reflection occurs. Here, assuming that a refractive index on a cover glass 2 side via the boundary surface between the cover glass 2 and specimen 3 is n1, and a refractive index on a specimen 3 side is n2, a critical angle θc is represented by the following equation (2).sin θc=n2/n1  (2)
Therefore, conditions of the incidence angle θ1 for realizing the total internal reflection illumination is represented by the following equation (3).n1·sin θ1>n2  (3)
On the other hand, to incline an incident light of the laser beam 5 passing through the objective lens 1 as in the Jpn. Pat. No. 3093145 shown in FIGS. 17A to 17C, a maximum incidence angle θmax that can be set depends on the numerical aperture (NA) of the objective lens 1, and is represented by the following equation (4).n1·sin θmax=NA  (4)
Therefore, for the conditions for realizing the total internal reflection illumination, the NA of the objective lens 1 needs to be larger than the refractive index n2.
In general, a refractive index of a living cell is about 1.37 to 1.38. The NA of the objective lens 1 for use needs to be about 1.4 at minimum.
At present, a magnification of the objective lens having an NA of 1.4 or more is limited to a high magnification of 60 times or more. To realize a high NA by the objective lens 1 having a low magnification, an effective diameter of the objective lens 1 needs to be increased. However, it is difficult to increase the effective diameter of the objective lens 1 while keeping a standard diameter of an attaching screw of the objective lens 1. Therefore, in the TIRFM shown in FIGS. 17A to 17C, total internal reflection fluorescence observation at a magnification of about 20 or 40 times is impossible.
In the TIRFM shown in FIG. 18, when the laser beam 5 is incident from a condenser lens for transmission illuminating 8 side, an illuminative range can be set without depending on the objective lens 1. Accordingly, the total internal reflection fluorescence observation using the objective lens 1 having a low magnification is possible.
However, in the TIRFM shown in FIG. 18, the laser illuminating device 7 is disposed right beside the point ball lens 9 of the condenser lens for transmission illuminating 8. Additionally, the laser illuminating device 7 itself needs to be rotated. Therefore, a considerable space is necessary including a holding section of a rotary mechanism and a space of a track of the rotating laser illuminating device 7. Consequently, a space in which the specimen 3 is laid is compressed. It is supposed that operation properties are remarkably impaired.
An irradiation range of the laser beam is set in such a manner that an observation range of the objective lens 1 having the low magnification can be illuminated. Then, in the observation with the objective lens 1 having the high magnification, only a part of the irradiation range of the laser beam is observed. Therefore, the laser beam with which another part is irradiated is useless.
An energy density of the laser beam on the surface of the specimen 3 is in inverse proportion to an irradiation area. In the observation with the objective lens 1 having the high magnification, the irradiation range of the laser beam is condensed so as to illuminate only a range required for the observation, and the energy density of the laser beam is preferably enhanced.
Especially, there is an experiment for the purpose of detection of very weak fluorescence such as a single molecule. In this experiment, the irradiation energy density of the laser beam is required to be as high as possible. On the other hand, in the TIRFM, the leak-out depth of the evanescent light is changeable. In recent years, the TIRFM has been spread in the site of the biological research. Furthermore, there has started to be a demand for the simultaneous illuminating of a plurality of optional wavelengths in optional depths.
This background has the following actual circumstances. Improvement of fluorescent protein such as GFP has been advanced, and it becomes easy to observe a dynamic state or a function of the living cell with multicolored fluorescence. Moreover, as seen also from Axelrod formula (equation (1)), the leak-out depth of the evanescent light depends also on the wavelength of the light. Therefore, there is a principle problem that a range to be observed differs, when the wavelength differs even at the equal laser beam incidence angle. There is also a realistic problem that a depth position of a tissue in a cell corresponding to each wavelength differs.
In the TIRFM, it is possible to switch the incidence angle or the wavelength of the laser beam at a high speed using mechanical means or electric driving means such as a motor. However, in cases where a simultaneous property in a strict meaning is required such as a case where a fast phenomenon is traced, there is a restriction on a high-speed switch. In this case, it is necessary to simultaneously-illuminate introductory portions of the laser beams disposed in a plurality of places.
However, in the TIRFM shown in FIG. 17A, a dichroic mirror is used in order to reflect the laser beam on a objective lens 1 side and to transmit the fluorescence on an observation side.
Additionally, when there are a plurality of wavelengths to be illuminated, the dichroic mirror needs to have wavelength characteristics of the corresponding multi-band. The dichroic mirror of the multi-band has a high difficulty in manufacturing, and is expensive. Furthermore, the dichroic mirror of the multi band has a bad separation level of the wavelength, and brightness and SN ratio of a fluorescent image are deteriorated. When the illuminative wavelengths are to be further increased halfway, the dichroic mirror needs to be newly prepared again.
On the other hand, to prevent the dichroic mirror from being used, as shown in FIG. 19, it is possible to dispose a total internal reflection mirror 12 in a position of an outermost portion of a pupil of the objective lens 1. However, it is necessary to dispose another total internal reflection mirror 13 also in the outermost portion of the pupil of the objective lens 1 on an opposite side in order to prevent the laser beam 5 totally reflected by the boundary surface between the cover glass and the specimen 3 from passing on an observation side.
Therefore, a considerable part of the pupil of the objective lens 1, which should have been originally used 100% for observation, is lost by the respective total internal reflection mirrors 12, 13. Therefore, a capability of the objective lens 1 is deteriorated.
In the TIRFM shown in FIG. 18, as described above, the laser illuminating device 7 is disposed right beside the point ball lens 9 of the condenser lens for transmission illuminating 8, and additionally the laser illuminating device 7 itself needs to be rotated. Therefore, a considerable space is required including the space of the track of the holding section of the rotation mechanism or the rotating laser illuminating device 7. In this constitution, when the laser illuminating device 7 including an emission angle adjustment section of independent laser beams is to be disposed, two laser illuminating devices at maximum can be disposed on opposite sides of the condenser lens for transmission illuminating 8.
Moreover, each TIRFM has a common problem. There is a case where a plurality of wavelengths are observed by the use of the evanescent lights having different depths. For example, when a shallow region is observed with B excitation, and a deep region is observed with G excitation, the B and G excitations can be simultaneously observed only in the shallow region. In this case, the image of the G excitation can only be observed as a defocused background image. Therefore, for example, the whole cell film is dyed by a fluorescent reagent, and the image of the TIRFM in the deep region can be used only in limited applications such as grasping of an approximate size of the cell.
While the objective lens is fixed, the surfaces in the different depths can be simultaneously observed. Then, the application of multi-wavelength TIRFM can further be broadened to simultaneous observation of forms of small organs in the vicinity of the cell film and inside the cell.